Table Of ContentReference: Hint. Hull. 1%: 45-51. (February. ll)99)
Calcium Dependence of Settlement and Nematocyst
Discharge in Actinulae of the Hydroid
Tubularia mesembryanthemum
SATORU KAWAII1 KEIJI YAMASHITA2 MITSUYO NAKAI. MIYUKI TAKAHASHI,
. ,
AND NOBUHIRO FUSETANI3'*
Biofouling Project, ERATO. Research Development Corporation ofJapan, c/o Yokohama R&D center,
Niigata Engineering Co. Ltd., 27 Shin-isogo-cho, Isogo-ku, Yokohama, 235. Japan
Abstract. The influence ofCa2+ and Mg2+ ions on both ameansofdispersal, arecomposedofaconical manubrium,
atrichous isorhiza (AI)dischargeand settlementofactinular oral- and aboral tentacles, and a basal protrusion that later
larvae of the hydroid Tubularia mesembryanthemum was becomes the settlement organ. Settlement-competent ac-
investigated. Mg2+-supplemented artificial seawater(ASW) tinulae initiate settlement behavior by contact ofthe aboral
completely inhibited both events at a concentration of 206 tentacle tip (ATT) with the substratum surface, which trig-
2+
mM, whereas loweredMg concentrations enhanced them. gers discharge of atrichous isorhiza (AI), the agglutinant
Ca2+ ions in the bathing solution highly regulated AI dis- nematocysts located at the ATT.
2+
charge and settlement, and Mg ions may down-regulate Upon reception of appropriate stimuli, nematocysts can
tesrheset,stelienemcvelenuntdtiswn.agsTGhiden3he+ifbfiaetncetddoLbfya3iCn+oo.r2wg+aan,siNcail2Cs+ao,2e+Cx-dacm2hi+ann,enLdea.l3+Lbal,roavcnakl-d et1ji9eo4cn2t)r.aeqdTuhiwersoeisvcelbaotstushbeusmleoecfshca(hTneaimrcodaerlnetca,enpd1t9co8hr8es)m.wieNcraeelmaistdteoinmctuyiltfiiee(dPeaxincntiittnah,-e
Gd3+. with half inhibitory concentrations (IC50) of 5800, tentacle ofthe sea anemone Aiptasia pallida: one sensitive
260, 53, 45. and 7 ju,M respectively; AI discharge was also to low molecular weight amino/imino compounds, and the
inhibited by these ions, with IC50 values of 6600, 500, 78. other specific for N-acetylated sugars (Thorington and
4i1n.voalnvdem5en^tMo.frsetsrpeetccthi-vaecltyi.vatTehdesCea2r+escuhltasnnseulgsgeisntthpeosssiigbnlale Htoersssianrgeeir,nvo19l8v8ead).inItnraidgdgietriionng,ntewmoattoycpyesstofdimsecchahragneo;reocneepi-s
transmission of both AI discharge and larval settlement. contact-sensitive (Thorington and Hessinger, 1988b) and
the other is tunable, vibration- and frequency-sensitive
Introduction
(Watson and Hessinger, 1989, 1991).
The colonial marine hydroid Tubularia mesembryanthe- The necessity of external Ca2+ ions for nematocyst dis-
mum Allman. 1871 (Hydrozoa: Tubulariidae) is a prolific charge has been demonstrated in Aiptasia mutabilis (San-
marine fouling organism that frequently dominates artificial toro and Salleo, 1991b), Pelagia noctiluca (Salleo et al.,
substrata used in aquaculture. Actinula larvae, produced as 1994a), Calliactisparasitica (Salleo etal.. 1994b), Chaiyb-
dea rastonii (Yanagita. 1973), and Anihopleura elegan-
Received 3 February 1998: accepted 19 October 1998. tissima (McKay and Anderson, 1988). In Hydra vulgaris,
+
1 Presentaddress:Natl.Inst.FruitSci..Min.Agric.Forest.Fish..Okitsu, discharge ofstenotele is regulated by a voltage- and Ca"
Shi2mPirzeus.enSthiazdudoreksas:424H-i0m2e0j4i.KJiaspahno.CEo-.maiLlt:[email protected]. dependent mechanism, allowing Ca2+ influx from the bath-
Hy3ogProes6e7n2t.aJdadpraens.s: Laboratory of Aquatic Natural Products Chemistry. cienlglusloalrutiCoan2(+Gititoenrsetinal.t,he199m4e)t.aTmohrepihnovsoilsveomfenhtyodfroizntoraan-
Graduate School of Agricultural and Life Sciences, The University of larvae has also been suggested on the basis of studies
Tok*yAou.thBournktyoo-wkhuo.mTockoryroes1p1o3n.deJanpcaen.should be addressed. monitoring the emission of bioluminescence from photo-
Abbreviations: AI, atrichous isorhiza: ATT. aboral tentacle tip. proteins, thereby visualizing changes in [Ca~ ]i during
45
KAWAII ET AL
46 S.
planular metamorphosis (Freeman and Ridgway, 1987, phore, and 24-h-old actinulae were used in the following
experiments unless otherwise stated.
1990).
Previous work ha> shown that Ca2+ release from intra-
cellular stores tri.^ered AI discharge in Tubularia mesem- Settlement assay
bryanthemum actinulae, followed by the inflow of Ca2+ Actinular settlement was assayed using six-well polysty-
eiptcilolianannnvarusiloil,elfafdyrvr1eo9tbsmm9heye7etnt)tht.slreieoFnolbmuufeaeronttuCthohsifaenar2gmCnm+oados-vro2melem+eu,emdttieiiAaaoonItnmntseodidrinoisptfncshohiottgaashhncrieeatgsliennaeubwtwlmoraaaarasrsatnlosusdcsseutyuutcesastntlptltileseaoycmcnt(elaeneKdc.tian.,cwToaaTwhmice-ioe- crpsmeoelitnnactetcrlaeeoipddslncaiaotoncnepgtseia6n(nuaClfmotaolerrerbnoiiwt3fnaa0glsAhSCs.cehoWlEaulkaneactWrnheeddlaelt5xus.pn2a1edcCretoirirmnnaeuiln2bantCeig..n,woaTcNTshuYhle)epa,reprlndeafuiatomsecrsbshmeeecwrwtdeeirlooielnfn
examined AI discharge and larval settlement using intact triTploicadtee.termine the reversibility of actinular settlement
tliaanirfnvliauneegnctvheaartoifowuesdriec-obnacanetdnhterdtaritivinaolnaersnttiofficcCaitaali2os+nseaawnaadntdMergC'(a+A2+Si-oWnc)sh.acnTonhnee-l lutahperovnwaeelrtlehapwtlearwceeermweeanlstahregodeflbyytshisenekvtieenrsgatloArreSaptlWtaaccehwmiinetgnhttsoreotghfuetlhaberotstAuroSfmWaco.ef
bSlaolclekoeresl(aYla,ng19a94nad,Sba;chGsi,tt1e9r8e9t;aSla.nt1o9r9o4)anwdasSalallesoo,e1x9a9m1-; oofftthheetteessttAASSWWwiwtahsreegxuclhaarnAgeSdWwi(trhouegahclhy hraelpflatcheemveontl)u.me
ined.
Larval behavior and atnchous isorhiza discharge
Materials and Methods
Each actinula was independently examined for the effect
Reagents of Ca2+, Mg2+. or Ca2+-channel blockers on its behavior
when an ATTwascontactedwith aclean micropipette oron
Reagent grade artificial seawater (ASW) consisting of AI discharge. Actinulae in regular ASW were washed and
NaCl (460 mM), KC1 (10.1 mM). CaCK (9.2 mM), and suspended in test ASWs containing various concentrations
MgCU (42.6 mM) at pH 7.6 was adjusted by addition of 5 of Ca2+ Mg2+ or Ca2+-channel blockers. Each actinula
mM imidazole. The concentration of Ca2+ was reduced, was held,by suct,ion applied through a micropipette attached
Mwigth2o+utacaclotredriinngglyt.heMogsm2o+l-asruiptpyleomfenthteedAASWS,Wbwyitahdj5u9s,ti7n5g, ttoo AthTeTsicdoentoafctthewiatdhheascilveeanprmoitcrruospiionp.etBteehwaeviroerealxarmeisnpeondsoens
125, and 206 mM of Mg2+ was prepared by mixing the suction-held actinula whose ATTs did not contact any sub-
regularASW with 370 mM MgCU aqueous solution (which strata.
is isotonic to ASW) in the ratios of 1:19, 1:9. 1:3, and 1:1. For monitoring AI discharge, an ATT of a suction-held
respectively, to compensate for the osmolarity. In the case actinula was immobilized and attached to the bottom of a
oNfaC+a. 2A+l-lchaCnan2e+l-bclhoacnkneerls.btlhoeckoesrmsolwareirtey wpuarschaadsjeudstefdrboym pmeitcrriopdiipsehttebyatatpapclhyeidngto gtehnetl"ewrissutc,t"iownhitchhroiusghtheapasretcobned-
Wako Pure Chemical Industries (Osaka, Japan). tween an ATT and an aboral tentacle. AI discharge of the
immobilized ATT was then triggered by the addition of 100
Biological materials jul ofASW thatcontained 200 mM K+ ions (K+-AASSW)Wand
had been adjusted to the osmolarity of regular by
Mature Tubularia mesembryanthemum colonies were reducing the Na+ concentration. AI discharge was observed
collected mainly from submerged fisheries nets and ropes in through the 40 X objective ofa microscope, andthe number
the vicinity of Nagai Port in Sagami Bay (eastern Japan, of AI discharged before the K+-ASW application (usually
13930'E. 3170'N). Colonies were divided into male and 0-1 was subtracted from the final total.
female, and were maintained separately with sand-filtered To) avoid larval disintegration in Ca2+-free ASW, a mi-
retunaln.in(g199s7e)a.waAtfetreratfe1rt6ilizati2onC, barsandcehsecrsiobfedfebmyalYeacmoalsohniiteas ctrhoopriopuegthtley-hweladshaecdtinaunldasiunspreegnudleadr iAnSCWa^2+w-afsreequAicSkWl.y aInmd-
with polyps bearing many actinulae were placed in filtered mediately afterward, an ATT of the actinula was immobi-
seawater (FSW; pore size. 0.45 ju,m). and sinking actinulae li/.ed as described above and AI discharge was triggered.
were collected. A single polyp could produce up to 300
4C
actinulae, which were maintained at prior to use in an [Ca:+ measurement
echdxooepnfuetirransiiendmtieonansgtr.etbcieTomavhekeeerfroanlaclontoridwmniuamnllagaienrrteewaslepieroanseneesditfvarertanon2ems1sfset.rhreeLd2maarCttvoearlfanornaalgsAeegvSoewnWraoa-sl- hlaaed[ACbaTe2]eT,+n],intwraweahstiemcdehawAsiItuhrdeitdhsecihnCarwagh2eo+lw-ecahsmeloiauntndituncsgedfo.lfuTolrihveeisncagecntaticntiuinlnduai-e-
CA2+ DEPENDENCE OF HYDROID SETTLEMENT 47
cator fura-2 (Kawaii el til.. 1997). The fluorescence inten-
sities of ATTs from fura-2-loaded actinulae were approxi-
mately 100 times stronger than those from non-labeled
actinulae, and were strongenough for [Ca2^]; measurement.
The value of the fluorescence ratio between excitation at
340 nm and 380 nm (Rwi/w,) was used to indicate [Ca2+]j.
The minimum interval was 1.9 s for ratiometric imaging.
Results
Ca2+ dependence ofactinular settlement
Reducing the external Ca:+ concentration had a signifi- -''" '.. 1.1 mMCa2: 1
cant effect on actinular settlement, and the effect was com- 80 120 160 200
cpienothmiiptbaiivrteealdbylaeatntltaoogwoennroirzcmeoandlcebnyitnrMa4tg.i62o-+n9s.(2F(i7gm.0%M1A)C.a3S21e+t%,tlbaeutmte2.ni3tt mwwaaMss external Mg2* (mM)
CaR2a+i,si8n%g the1M0%g2a+t 1c.o1ncmenMtraCtai2o+n).inhibited actinular set-
tlement; 100% settlement was obtained at 75 mM Mg2+
50% at 125 mM Mg2+, and 6.7% 121+.6% at 206 mM. ,
The relative inhibitory effect of Mg on actinular set-
tlement was magnified at lower Ca2+. In ASW containing
2.3 mM Ca2+ settlement was reduced to 53% 12% at 59
mM Mg2+ and, completely inhibited at 206 mM Mg2+. The
effects ofCa2+ and Mg2+ concentration on settlement were
ANOVA
statistically significant as assessed by two-way
(Table I). Furthermore, there was a significant interaction
between Ca2+ and Mg2+. In each case the dose-dependent
reduction ofactinularsettlement was reversed by rinsing the
ASW
larvae with regular 9 h after the experiment.
40 80 120 160 200
+
Antagonistic effect ofMg~ external Mg2+ (mM)
2+
Raising the Mg concentration in an otherwise regular
AniAinoIuShnmidWbbciieostnrhcochareoyadnfrtegdarfeinafstewcicianhtohasnirobgfcwieotaMdmosprgAylIle2oet^wwfefealiersycnetcd4ar..ore0rnaIesnseAtdIeA4d.wSd2ahitWasetc2nhc10ao26trn5hgtemeamieM(MnxFitineMgMrg.gng~a4Il2.B+6+)C.;,maTath2nhMe+de cdcsaoephnenvadFcciriiaegMgftneiuitgeorridnae:nst+ci1ao.o(rinntnocisnefs=i(no.cAtif)ra5Mlac)etA.aisacloe(ntcnaBmissw)uuamlowtKfaeearr+ense-xdectAtoeotmnrSblatnteWaagamilnientenniCretsniadiggi:ugnb+medyr.ia.refVtfdaF=ieevfirraievtectrieniracatilaglcliacshrnooebvgnuaaacsrweetsanhtwiteiseernorrandrteiuhwicimioaiztbntnahseecru(dsobAitfaIofat)ffnCeedardddae~iirnssa+dt--t
CaCaAnnaaNdd22O++4w,..Va4AdAsIiscdrcoieh3smva.cepr1haglaleearettdgwe7ealt5yshwamatlisnoMthwhrieeebMrdieetgufedcfd2ee+tcdota.tst4Ioon.1f352A.5C7Sam2W1+M.34c.ao8naMntdatgta5Mi529ng9+im.n2mg+TMMw2c.ooM3Mn-gcgwme2a2nM+y+-, csrahtenaasdnrpdgeaeicrdntdciAdrvIeeevaliiasynt.eiedoaAncstd.hodia7tb.oi6roanl toe1fn.t6Maclgaen2dt+ip9-.r(2neduce110d.).9AVaetSrt2Wi5cal(a5nbdamrsM10)indmiacMlas,toe
tration on AI discharge were statistically signifi2c+ant, and th2a+t induced sinuous movement of the aboral tentacles.
there was a significant interaction between Ca and Mg
(Table I). Again, this dose-dependent reduction of AIASdiWs- Effects ofCa2+-channel blockers on actinular settlement
charge was reversed by rinsing the larvae with regular
2+
3 h after the experiment. The effectsofinorganic Ca -channel blockers on actinular
Figure 2 indicates significant enhancement of AI dis- settlementwereexaminedinvariousconcentrationsofchloride
charge in Mg2+-reduced ASW (one-way ANOVA; F - salt cations added to regular ASW. In each case, a dose-
1 1.9759, P < 0.0001). In this experiment. AI discharge was dependent reduction ofactinularsettlement wasobserved (Fig.
triggered by K+-ASW (100 mM). The number of dis- 3A). The following ions are listed in order of increasing
charged AI was 2.1 0.6 at 50 mM Mg2+ (regular ASW) inhibition efficiency: Co2+ < Ni2+ < Cd2+ < La3+ < Gd3+.
48 S. KAWAII ET AL.
Table I
Results oftwo-wayANOVA toassess the effects ofCo""1" andMg:*
concentrationson (AI iciinularsettlementand(B) atrichous isorhiza
(AI) discharge
Source*
CA2+ DEPENDENCE OF HYDROID SETTLEMENT 49
this is the first demonstration of Ca2+-dependent nemato-
cyst discharge in larvae. We also confirmed and quantified
the Ca2+-dependence of actinular settlement in Tubitlaria
mesembryanthemum. Reduced-Ca2+ seawater also inhibits
metamorphosis of Hydractinia cchintita plunula larvae
(Berking, 1988; Muller, 1985). The EC50 of external Ca2+
mM
was 3 for T. mesembryanthemum actinular settlement,
B
comparable to that of both H. echimita settlement and
actinularAI discharge (Kawaii etui, 1997). Since actinulae
immersed in Ca2+-free ASW tended to disintegrate, settle-
ment assays were not performed in Ca2+-free ASW.
The action of K+-ASW was strongly inhibited by in-
creasing the Mg2+ concentration of the bathing solution,
O 2+
CD and the inhibitory effects of the Mg ion increased when
the external Ca2+ concentrations were lowered. Similarly,
Muller (1985) found that TPA-induced metamorphosis of
cc 1.2 40 120 160 H. echimita was promoted in Mg2+-reduced seawater.
2.2 Mg2+ and Ca2+ ions compete with each other in many
biological processes; consequently, we expected that an
2+
increase of Mg concentration in the bathing solution
would reduce the Ca2+ influx, thus inhibiting actinular
settlement and AI discharge. However, the rise of R340/38o
values measured in Mg2+-supplemented ASW was equiva-
lent to that observed in Ca2+-free ASW, and there was no
1.6 influx ofCa2+ ions from the Mg2+-supplemented ASW to
nematocytes. These observations indicate that inhibition of
2+
AI discharge by Mg did not result from the lowering of
1.2 nanedmaCtaoc2y+teion[s.CaT2h+e],enlehvaenlcebmyenctomopfetAiItidviescihnafrlguexaonfdMlagrv2a+l
40 80 120 160 2+
settlement by Mg reduction may be a result of cation-
time (sec) mediated alteration ofmembrane-associated cell function in
2+
signal transduction, and Mg ions would therefore regulate
Ca^F-igfurreee4a.rtif|icCiaal2*s],eatwraatnseiren(tAsSdWu)r,ing(Ba)trMigch-o+us-siuspoprhliezmaendtiescdhaArSgeWin((2A0)0 AI discharge and settlement ofthe hydroid as an inhibitory
mM Mg2+), (C) 20 pMGd'+-ASW. and (D) regular ASW. element.
mented ASW (200 mM Mg2+), 20 yM Gd3+-ASW, and Involvement ofstretch-activated (SA) channels
regular ASW. Despite the discharge inhibition by Gd1+ ions, It is natural for us to speculate about involvement of
R340/380 transients were induced by the addition of K+-ASW stretch-activated (SA) channels in actinular settlement, be-
(200 mM). The rise of RJ^J/MO measured in the presence of cause the inhibitory effect of gadolinium ions at low con-
Gd3+ ions was similartothatobserved in Ca^-free ASWand centrations in ourstudy is comparable to that ofopening SA
ASW
considerably lowerthan thatobserved in regular [AR,4()/ channels in patch-clamped Xenopus oocytes (Yang and
380 = 0.23 0.1 1, 0.18 0.17, and 0.62 0.24, respective- Sachs, 1989). Gd3+ is the most effective blocker of SA
ly]. No significantdifference wasobservedbetweenCa2+-free channels found to date, although it also has some effect on
AmeSntWed(AA[SCaW2+(]0,.2=8 0.301.11, 0n.0=9,6)n. = 6) and Mg2+-supple- CaW2+ecphraenvnieolussl(ySdaedmoosnhsitmraateetdatt..hat19b9o2t)h. ATT contact with
substrata and chemical stimuli are necessary to induce AI
Discussion discharge (Kawaii et ai, 1997). By itself, a mechanical
Ca2^-dependence ofsettlement and atrichous isorhiza stimulus, such as immobilization of ATT by suction and
vibration applied through a clean micropipette, did not
discharge trigger AI discharge. However, contact with a bacterial-
Although external Ca2+ ions have been reported as being film-coated micropipette did trigger discharge and also in-
required for nematocyst discharge in several species of duced sinuous movement of the tentacles. Moreover, K+-
cnidaria (Salleo et ai, 1993; Santoro and Salleo, 1991a, b). ASW treatment did not result in AI discharge from ATTs
50 S. KAWAII ET AL
that were not in contact with any substrata. These results phosis. The inhibitory effect of the Ca2+-channel blockers
suggest that K+-ASW treatment replaced sensory input of demonstrated that AI discharge and subsequent signal trans-
+
chemical stimuli, but did not mimic physical stimuli from mission require involvement of Ca~ channels.
ATT contact. Therefore, the observation that the stretching
caused by immobilization of ATT did not trigger AI dis- Acknowledgments
charge does not rule out the involvement ofSA channels in
The authors thank Dr. E. Hunter (The Centre for Envi-
the discharge mechanism. &
ronment, Fisheries Aquaculture Science, Suffolk, U. K.)
Effect ofCa2+-channel blockers JaanpdanD)r.forC.theGi.r vSaaltuuaibtole(sJugAgPeAstNionNsU.S Co., Ltd., Tokyo,
All the Ca2+-channel blockers tested for inhibitory ef-
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ited AI discharge and caused slight elevation of R^o/sso- activation of in situ nematocytes in acontia of Calliactis parasitica.
2+
TtCohaseo2sl+.ersemlaelasle[fCraom],inttrraancseilleunltasrcsotuolrdesbteo innetmeraptroectyetdeascy-a SantEooxfrptoeh,reiGea.nc,toinatanida47oA:f.7ASi0ap1lt-la7es0oi.3a.1m9u9t1abb.ilisTihseadCiasc2h+a-rigneduocfeidnrseitsuponnesmea.tJo.cyEsxpt.s
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